1. Field of the Invention
This invention relates to enzyme analysis and more particularly to a method for enzyme analysis with aromatic amines coupled to aromatic aldehydes.
2. Description of the Prior Art
One of the principles most widely used for colorimetrically demonstrating the presence of hydrolytic enzymes in tissue sections and homogenates involves the two step process:
(1) substrate .sup.enzyme coupling component+ other products PA1 (2) coupling component+ dye forming agent .fwdarw. dye.
The coupling components are aromatic hydroxy compounds or amines, usually of the naphthalene series. The dye forming agents are typically diazonium salts or other compounds which react in a similar manner with the coupling component to form a dye. The dyes formed when diazonium salts are used are azo dyes. One of the coupling components which has been used in histochemical localization of enzymes is 4-methoxy-2-napthylamine which was first made by Rosenblatt, Nachlas and Seligman (Synthesis of m-Methoxynaphthylamines as precursors for Chromogenic Substrates, 80 Jour. Am. Chem. Soc. 2463, July 1958). It was first used histochemically for the localization of enzymes wherein only one amino acid, y-Glutamyl, was attached. A. Rutenburg et al., Histochemical and Ultrastructural Demonstration of y-Glutamyl Transpeptidase Activity, 17 Jour. Histochemistry and Cytochemistry 517, 1969.
Carbobenzoxydiglycyl- 1-arginyl-2-napthylamine hydrochloride was prepared by Plapinger et al. for the study of the enzyme trypsin. (Synthesis of Chromogenic Arginine Derivatives as Substrates for Trypsin, 30 Jour. of Organic Chem. 1871, June 1956). Carbobenzoxydiglycyl- 1-arginyl-2-napthylamide as a trypsin substrate was used by Nachlas, Plapinger and Seligman, (Role Of Some Structural Features of Substrates on Trypsin Activity, 108 Archives of Biochemistry and Biophysics 266, 1964). While this compound is desirable because of its capability of facilitating the study of trypsin-like enzymes, its slow coupling rate with certain diazonium salts and lack of strong color of the coupled compound is an awkward and unwanted drawback.
Many of the prior art processes for determining enzyme concentrations have been based on amino acid derivatives of 2-napthylamine. (Role of Some Structural Features of Substrates on Trypsin Activity, supra.) Use of the 2-napthylamine coupling component in conjunction with a dye forming agent presents an inconveniently slow and relatively insensitive method for determining enzyme concentrations. Alternatively, the 2-napthylamine coupling component can be exposed to ultraviolet light and its fluorescence measured directly. While this eliminates problems associated with the use of a dye forming agent, the frequency of fluorescence (blue) is difficult to measure with inexpensive instruments and is similar to other materials often present in material being assayed.
U.S. Pat. No. 3,862,011 to Smith describes a colorimetric method for determining enzyme concentration in homogenates such as serum or tissue extracts employing 4-methoxy-2-napthylamines. To the homogenate is added an amino acid derivative of 4-methoxy-2-napthylamine (4M2NA) which acts as a substrate for a specific enzyme depending on the particular amino acid residue attached to the 4M2NA molecule. Enzymatic hydrolysis cleaves the arylamine bond to yield free 4M2NA which is then measured by azo coupling to an appropriate azo dye and thereafter using absorption photometry. Although not set forth in U.S. Pat. No. 3,862,011, 4M2NA has been known to be highly fluorescent, but with the undesirable blue color.